The Hox-2.1 gene is one of homeobox-containing genes located in the Hox-2 cluster on mouse chromosome 11. In this study, we have examined transcription of the Hox-2.1 gene during differentiation of F9 embryonal carcinoma cells induced by treatment with retinoic acid. The level of Hox-2.1 mRNA increases rapidly after induction of differentiation and then falls. Nuclear run-on experiments demonstrate that the rate of transcription for the Hox-2.1 gene also increases upon differentiation. Treatment of F9 cells with a DNA topoisomerase II inhibitor etoposide (VP-16) during differentiation blocks the accumulation of Hox-2.1 mRNA. Nuclear run-on analyses reveal that etoposide inhibits transcription of the Hox-2.1 gene during F9 cell differentiation. Measurements of the level of Hox-2.1 mRNA after blocking transcription by actinomycin D show that etoposide does not affect stability of the mRNA. These observations indicate that DNA topoisomerase II is involved in the control of Hox-2.1 gene transcription.